Angiogenesis, the process of new blood vessel formation, is key to normal organ development, as well as to various pathological disorders like cancer, arthritis, diabetic retinopathy and restenosis (A. W. Griffloen et al., Biochem. J., 354, 233–242 (2001)). βPep-25, which has the sequence ANIKLSVQMKLFKRHLKWKIIVKLNDGRELSLD (SEQ ID NO:1), a designed cytokine-like β-sheet-forming peptide 33mer, is an anti-angiogenic compound being developed to combat these pathological disorders (K. H. Mayo et al., Angiogenesis, 4, 45–51 (2001); and S. Liekens et al., J. Biochem. Pharm., 61, 253–270 (2001)).
The use of agents that can inhibit angiogenesis, particularly in anti-tumor research (e.g., M. S. O'Reilly et al., Cell, 88, 277–285 (1997); and T. Boehm et al., Nature, 390, 404–407 (1997)), has indicated that anti-angiogenic therapy will be a promising therapeutic modality in the near future. To date, the search for angiogenic inhibitors has been focused on controlling two of the processes that promote angiogenesis: endothelial cell (EC) growth and adhesion (G. Molema et al., Immunol. Today, 19, 392–394 (1998); and J. Folkman et al., Nature Med., 1, 27–31 (1995). Targeting EC as an anti-tumor treatment is attractive primarily because EC are more accessible than are other cells to pharmacologic agents delivered via the blood, and EC are genetically stable and are not easily mutated into drug resistant variants.
Most anti-angiogenic agents have been discovered by identifying endogenous molecules, primarily proteins, which inhibit EC growth. This traditional approach has produced a number of anti-angiogenics, such as platelet factor-4 (PF4), thrombospondin, tumor necrosis factor-α (TNF-α, depending on its concentration), interferon-γ inducible protein-10, angiostatin, endostatin and vasostatin and bactericidal-permeability increasing (BPI) protein. See, for example, M. S. O'Reilly et al., Cell, 88, 277–285 (1997); S. K. Gupta et al., J. Cell Biol., 127, 1121–1127 (1994); S. S. Tolsma et al., J. Cell Biol., 122, 497–511 (1993); N. Sato et al., Japan Natl. Cancer Inst., 76, 1113–1121 (1986); V. J. Palombella et al., J. Biol. Chem., 264, 18128–18136 (1989); B. Robaye et al., Am. J. Pathol., 138, 447–453 (1991); A. D. Luster et al., J. Exp. Med., 182, 219–231 (1995); M. S. O'Reilly et al., Cell, 79, 315–328 (1994); S. E. Pike et al., J. Exp. Med., 188, 2349–2356 (1998); and D. W. J. Van der Schaft et al., Blood, 96, 176–181 (2000). About forty anti-angiogenic agents are currently known.
Most anti-angiogenic proteins are compositionally similar, having a relatively high incidence of hydrophobic and positively charged residues and are folded primarily as β-sheets (A. R. Mire Sluis et al., J. Immunol. Methods, 200, 1–16 (1997)): interleukin-1 (IL-1), tumor necrosis factor (TNF), lymphotoxin (LT or TNF-β), transforming growth factor-β (TGF-β), endostatin. See, for example, J. P. Priestle et al., Proc. Natl. Acad. Sci. USA, 86, 9667–9671 (1989); E. Y. Jones et al., Nature, 338, 225–228 (1989); M. J. Eck et al., J. Biol. Chem., 267, 2119–2122 (1992); S. Daopin et al., Science, 257, 369–373 (1992); and E. Hohenester et al., EMBO J., 17, 1656–1664 (1998).
Recently, a designed amphipathic β-sheet-forming peptide 33mer, βpep-25 was shown to be a potent inhibitor of EC growth and angiogenesis. βpep-25 is more effective at inhibiting EC growth than PF4 and several other well-known angiogenesis inhibitors such as angiostatin, endostatin, AGM-1470 and thrombospondin-1. βpep-25 is believed to act by specifically blocking adhesion and migration of angiogenically-activated EC, leading to apoptosis and ultimately to inhibition of angiogenesis in vitro and in vivo and inhibits tumor growth by up to 80% in various models. See, for example, D. W. J. Van der Schaft et al., Faseb J., 16, 1991–1993 (2002); and R. P. Dings et al., Cancer Res., 63, 382–385 (2003).
For the smart design of smaller compounds, the identification of specific amino acid residues and their spatial relationships are used. One of the main goals among structural biologists and pharmaceutical chemists is to develop small molecules and potentially more effective anti-tumor agents. Nevertheless, for anti-angiogenic proteins such structure-activity relationships (SAR), i.e., specific residues and conformations which impart activity, are sorely needed, and even the analysis of high-resolution molecular structures of a number of anti-angiogenic proteins, e.g., endostatin, PF4, and BPI, has yet to provide this information. See, for example, E. Hohenester et al., EMBO J., 17, 1656–1664 (1998); K. H. Mayo et al., Biochemistry, 34, 11399–11409 (1995); and L. J. Beamer et al., Science, 276, 1861–1864 (1997).